Methods for increasing mannose content of recombinant proteins

ABSTRACT

The present invention relates to methods of upregulating the high mannose glycoform content of a recombinant protein during a mammalian cell culture by manipulating the mannose to total hexose ratio in the cell culture media formulation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/121,419, filed on Dec. 14, 2020, which is a continuation of U.S.patent application Ser. No. 16/537,490, filed on Aug. 9, 2019, now U.S.Pat. No. 10,894,972, which is a continuation of U.S. patent applicationSer. No. 16/039,123, filed on Jul. 18, 2018, now U.S. Pat. No.10,421,987, which is continuation of U.S. patent application Ser. No.15/285,392, filed on Oct. 4, 2016, now U.S. Pat. No. 10,184,143, whichis a divisional of U.S. patent application Ser. No. 14/289,943, filedMay 29, 2014, now U.S. Pat. No. 9,481,901, which claims the benefit ofU.S. Provisional Application 61/828,969, filed May 30, 2013, thecontents of which are incorporated herein by reference in theirentireties.

BACKGROUND OF INVENTION

Glycoforms of a protein expressed by Chinese hamster ovary (CHO) hostcell are largely determined during cell line generation and cloneselection. However, high mannose glycoform content can also be affectedby cell culture conditions (Pacis et al., (2011) Biotechnol Bioeng 108,2348-2358). Proteins produced in mammalian cell cultures may containvaried levels of high mannose glycoforms such as Mannose5 (Man5),Mannose6 (Man6), Mannose7 (Man7), Mannose8 (Man8) and Mannose9 (Man9).High mannose glycoform content of therapeutic proteins is a criticalquality attribute that has been found to affect pharmacokineticproperties of certain therapeutic antibodies (Goetze, et al., (2011)Glycobiology 21, 949-59; Yu, et al., 2012) MAbs 4, 475-87).

It is common in therapeutic protein industry to seek a desired range ofhigh mannose glycoform content for a protein product due to processchanges, scale-up, improvements or the need to match existing antibodyquality attributes. Methods for manipulating high mannose glycoformcontent of a protein in cell culture include changes in mediacompositions, osmolality, pH, temperature, etc (Yu, et al., supra, Paciset al., supra, Chee Furng Wong et al., (2005) Biotechnol Bioeng 89,164-177; Ahn, et al., (2008) Biotechnol Bioeng 101, 1234-44). Lowertemperatures, such as below 32° C., have been shown to increase highmannose glycoforms and reduce antennary structure and sialylation ofrecombinant erythropoietin (Ahn et al. (2008) Biotechnol Bioeng 101,1234-44). Recently, Pacis and colleagues demonstrated that antibody Man5level can be increased more than two-fold from 12% to 28% by increasingmedium osmolarity and culture duration time (Pacis et al., supra). Theeffectiveness of these methods is specific to cell lines, molecule typesand media environment and is typically obtained by trial and error.Additionally, these methods tend to also alter antibody productivity,cell culture behavior and other antibody quality attributes.

Therefore there is a need for a method for predictably manipulating highmannose glycoform regulation. Such a method would benefit the processdevelopment of therapeutic proteins. The invention provides a methodthat regulates high mannose glycoform content by manipulating themannose to total hexose ratio in cell culture media.

SUMMARY OF THE INVENTION

The present invention provides a cell culture media containing mannose,wherein the mannose to total hexose ratio in the cell culture media isgreater than 0 but less than 1.0. In one embodiment the mannose to totalhexose ratio in the cell culture media is greater than or equal to 0.25.In one embodiment the mannose to total hexose ratio in the cell culturemedia is greater than or equal to 0.5. In another embodiment the mannoseto total hexose ratio in the cell culture media is greater than or equalto 0.75. In another embodiment the mannose to total hexose ratio in thecell culture media is less than or equal to 0.94. In another embodimentthe cell culture media contains at least 3 g/L mannose. In anotherembodiment the cell culture media contains at least 6 g/L mannose. Inanother related embodiment the cell culture media contains at least 9g/L mannose. In another embodiment the cell culture media contains atleast 11.25 g/L mannose. In another embodiment is provided use of a cellculture media described above for upregulating the high mannoseglycoform content of a recombinant protein during a mammalian cellculture process.

The present invention provides a method for upregulating the highmannose glycoform content of a recombinant protein during a mammaliancell culture process comprising; establishing a mammalian cell culturein a bioreactor with a cell culture media that does not contain mannose;and maintaining the cell culture with a cell culture media containingmannose, wherein the mannose to total hexose ratio in the cell culturemedia is greater than 0 but less than 1.0. In one embodiment the cellculture is maintained by perfusion. In one embodiment perfusion beginson or about day 3 to on or about day 9 of the cell culture. In a relatedembodiment perfusion begins on or about day 3 to on or about day 7 ofthe cell culture. In one embodiment perfusion begins when the cells havereached a production phase. In one embodiment perfusion comprisescontinuous perfusion. In one embodiment the rate of perfusion isconstant. In one embodiment perfusion is performed at a rate of lessthan or equal to 1.0 working volumes per day. In one embodiment the cellculture receives bolus cell culture media feeds prior to days 3-7 of theculture. In one embodiment the mammalian cell culture is established byinoculating the bioreactor with at least 0.5×10⁶ to 3.0×10⁶ cells/mL ina serum-free culture media. In one embodiment the mammalian cell cultureis established by inoculating the bioreactor with at least 0.5×10⁶ to1.5×10⁶ cells/mL in a serum-free culture media. In one embodiment theinvention further comprises a temperature shift from 36° C. to 31° C. Inone embodiment the invention further comprises a temperature shift isfrom 36° C. to 33° C. In a related embodiment the temperature shiftoccurs at the transition between the growth phase and production phase.In a related embodiment the temperature shift occurs during theproduction phase. In another embodiment the invention further comprisesinducing cell growth-arrest by L-asparagine starvation followed byperfusion with a serum-free perfusion media having an L-asparagineconcentration of 5 mM or less. In another embodiment the inventionfurther comprises inducing cell growth-arrest by perfusion with aserum-free perfusion media having an L-asparagine concentration of 5 mMor less. In a related embodiment the concentration of L-asparagine inthe serum-free perfusion media is less than or equal to 5 mM. In arelated embodiment the concentration of L-asparagine in the serum-freeperfusion media is less than or equal to 4.0 mM. In a related embodimentthe concentration of L-asparagine in the serum-free perfusion media isless than or equal to 3.0 mM. In a related embodiment the concentrationof L-asparagine in the serum-free perfusion media is less than or equalto 2.0 mM. In a related embodiment the concentration of L-asparagine inthe serum-free perfusion media is less than or equal to 1.0 mM. In arelated embodiment the concentration of L-asparagine in the serum-freeperfusion media is 0 mM. In a related embodiment the L-asparagineconcentration of the cell culture media is monitored prior to and duringL-asparagine starvation. In yet another embodiment the inventioncomprises that the packed cell volume during a production phase is lessthan or equal to 35%. In a related embodiment the packed cell volume isless than or equal to 35%. In a related embodiment the packed cellvolume is less than or equal to 30%. In a related embodiment the viablecell density of the mammalian cell culture at a packed cell volume lessthan or equal to 35% is 10×10⁶ viable cells/ml to 80×10⁶ viablecells/ml. In another embodiment he viable cell density of the mammaliancell culture is 20×10⁶ viable cells/ml to 30×10⁶ viable cells/ml. Inanother embodiment the perfusion is accomplished by alternatingtangential flow. In a related embodiment the perfusion is accomplishedby alternating tangential flow using an ultrafilter or a microfilter. Inyet another embodiment the bioreactor has a capacity of at least 500 L.In yet another embodiment the bioreactor has a capacity of at least 500L to 2000 L. In yet another embodiment the bioreactor has a capacity ofat least 1000 L to 2000 L. In yet another embodiment the mammalian cellsare Chinese Hamster Ovary (CHO) cells. In yet another embodiment therecombinant protein is selected from the group consisting of a humanantibody, a humanized antibody, a chimeric antibody, a recombinantfusion protein, or a cytokine. In yet another embodiment the inventionfurther comprises a step of harvesting the recombinant protein producedby the cell culture. In a further embodiment the recombinant proteinproduced by the cell culture is purified and formulated in apharmaceutically acceptable formulation. In a further embodiment thehigh mannose glycoform content of a recombinant protein is increasedcompared to a culture where the cells are not subjected to a cellculture media containing a mannose to total hexose ratio of greater than0 and less than 1.0.

The present invention provides a method for upregulating the highmannose glycoform content of a recombinant protein during a mammaliancell culture process comprising; establishing a mammalian cell culturein a bioreactor with a cell culture media that does not contain mannose;growing the mammalian cells during a growth phase with a cell culturemedia that does not contain mannose; initiating and maintaining aproduction phase in the cell culture by perfusion with a serum-freeperfusion media containing mannose, wherein the mannose to total hexoseratio in the perfusion media is greater than 0 but less than 1.0.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows a graph of effects of different carbon sources on viablecell density.

FIG. 1B shows a graph of effects of different carbon sources onviability.

FIG. 1C shows a graph of effects of different carbon sources on antibodyproduction.

FIG. 1D shows a graph of effects of different carbon sources on % HM.

FIG. 2A shows a graph showing that titration of glucose by mannose inthe media correlates with high mannose (HM) upregulation withoutimpacting cell growth and antibody production. % HM.

FIG. 2B shows a graph showing that titration of glucose by mannose inthe media correlates with high mannose (HM) upregulation withoutimpacting cell growth and antibody production. Viable cell density.

FIG. 2C shows a graph showing that titration of glucose by mannose inthe media correlates with high mannose (HM) upregulation withoutimpacting cell growth and antibody production. Viability.

FIG. 2D shows a graph showing that titration of glucose by mannose inthe media correlates with high mannose (HM) upregulation withoutimpacting cell growth and antibody production. Antibody titer.

FIG. 3A shows a graph that demonstrates that high mannose (HM)modulation effect was universal and predictable across CHO cell linesexpressing different antibodies (Cell line A).

FIG. 3B shows a graph that demonstrates that high mannose (HM)modulation effect was universal and predictable across CHO cell linesexpressing different antibodies. (Cell line B).

FIG. 3C shows a graph that demonstrates that high mannose (HM)modulation effect was universal and predictable across CHO cell linesexpressing different antibodies (Cell line C).

FIG. 3D shows a graph that demonstrates that high mannose (HM)modulation effect was universal and predictable across CHO cell linesexpressing different antibodies (Cell line D).

FIG. 3E shows a graph that demonstrates that high mannose (HM)modulation effect was universal and predictable across CHO cell linesexpressing different antibodies (Cell line E).

FIG. 4A shows a graph showing that high mannose (HM) modulation effectcan be scaled up and maintain equivalent cell growth, viability andantibody titer using cell culture media having different mannose tototal hexose (M/H) ratios. Viable cell density.

FIG. 4B shows a graph showing that high mannose (HM) modulation effectcan be scaled up and maintain equivalent cell growth, viability andantibody titer using cell culture media having different mannose tototal hexose (M/H) ratios. Viability.

FIG. 4C shows a graph showing that high mannose (HM) modulation effectcan be scaled up and maintain equivalent cell growth, viability andantibody titer using cell culture media having different mannose tototal hexose (M/H) ratios. Antibody titer.

FIG. 4D shows a graph showing that high mannose (HM) modulation effectcan be scaled up and maintain equivalent cell growth, viability andantibody titer using cell culture media having different mannose tototal hexose (M/H) ratios. % HM.

FIG. 5A shows a graph of metabolic profiles of three bioreactors withdifferent mannose to total hexose ratios (M/H ratios) in the media.Glucose levels lower than 1 g/L may not be accurately measured byNovaFlex. Lactate

FIG. 5B. Shows a graph of metabolic profiles of three bioreactors withdifferent mannose to total hexose ratios (M/H ratios) in the media.Glucose levels lower than 1 g/L may not be accurately measured byNovaFlex. Ammonia.

FIG. 5C. Shows a graph of metabolic profiles of three bioreactors withdifferent mannose to total hexose ratios (M/H ratios) in the media.Glucose levels lower than 1 g/L may not be accurately measured byNovaFlex. Glucose.

FIG. 5D. Shows a graph of metabolic profiles of three bioreactors withdifferent mannose to total hexose ratios (M/H ratios) in the media.Glucose levels lower than 1 g/L may not be accurately measured byNovaFlex. Glutamine.

FIG. 6. Shows a good correlation was demonstrated between mock perfusionsamples and bioreactor samples regarding the high mannose (HM)modulation effect and its predictability with different mannose to totalhexose ratios (M/H ratio). Square: mock perfusion; Circle: bioreactor.

FIG. 7. Possible mechanisms contributing to the high mannoseupregulation in high mannose to total hexose ratio media. Threepathways, including GDP-mannose biosynthesis, early glycosylation andGluNAc biosynthesis, may involve in increased accumulation of differenthigh mannose species.

DETAILED DESCRIPTION OF THE INVENTION

High-mannose glycoforms (HM) are increasingly recognized as criticalquality attributes for therapeutic protein products. Previous attemptsto control high mannose glycoform content have been essentiallyempirical and it has been challenging to target desired high-mannosecontent during recombinant protein process development. As describedherein, the high mannose glycoform content on recombinant proteins maybe controlled by manipulating the ratio of mannose to total hexose inthe cell culture media. Such manipulation was found to be predictive,scalable, linear and applicable to different cell lines, making thismethod a powerful tool for upregulating the percent of high mannoseglycoforms in a more predictable manner. The inventive method preciselycontrols the percent of high mannose glycoforms of a recombinant proteinwithout compromising cell growth and titer.

The present invention provides a cell culture media containing mannose,wherein the mannose to total hexose ratio in the cell culture media isgreater than 0 but less than 1.0 and use of such cell culture media forupregulating the high mannose glycoform content of a recombinant proteinduring a mammalian cell culture process. As demonstrated herein,advantage may be taken of the correlation between high mannose glycoformcontent on recombinant proteins and the mannose to total hexose ratio inthe cell culture media. Modulating glycoform content in such a manner isa powerful tool to manipulate a critical attribute of a therapeuticprotein.

The invention provides a method for upregulating high mannose glycoformsto achieve desired product quality attributes while maintainingdesirable levels of certain cell culture parameters such as volumetricproductivity, cell viability, and/or density. The method makes us of acell culture media wherein the mannose to total hexose ratio is between0 and 1.10. Also provided is a method for upregulating the high mannoseglycoform content of a recombinant protein during a mammalian cellculture process comprising; establishing a mammalian cell culture in abioreactor with a cell culture media that does not contain mannose; andmaintaining the cell culture with a cell culture media containingmannose, wherein the mannose to total hexose ratio in the cell culturemedia is greater than 0 but less than 1.0.

As used herein, high mannose glycoforms (HM, usually provided as apercentage) refers to a protein having one or more high mannose glucansattached. High mannose glycans include Mannose-5, Mannose-6, Mannose-7,Mannose-8 and Mannose-9.

Mannose to total hexose ratio (M/H) is determined by dividing the amountof hexose (including mannose) by the amount of mannose in the liquidmedia that is fed into the bioreactor. For example, if the mediacontains 6 g/L mannose and 6 g/L glucose, the M/H ratio is 0.5. Hexoserefers to a monosaccharide having 6 carbon atoms per molecule, such asmannose, glucose, galactose and fructose.

Carbohydrate moieties are described herein with reference to commonlyused nomenclature for oligosaccharides. A review of carbohydratechemistry which uses this nomenclature can be found, for example, inHubbard and Ivatt, Ann. Rev. Biochem. 50:555-583 (1981).

For the purposes of this invention, cell culture medium is a mediumsuitable for growth of animal cells, such as mammalian cells, in invitro cell culture. Cell culture media formulations are well known inthe art. Typically, cell culture media are comprised of buffers, salts,carbohydrates, amino acids, vitamins and trace essential elements.Various tissue culture media, including defined culture media, arecommercially available, for example, any one or a combination of thefollowing cell culture media can be used: RPMI-1640 Medium, RPMI-1641Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum EssentialMedium Eagle, F-12K Medium, Ham's F12 Medium, Iscove's ModifiedDulbecco's Medium, McCoy's 5A Medium, Leibovitz's L-15 Medium, andserum-free media such as EX-CELL™ 300 Series (JRH Biosciences, Lenexa,Kans.), among others. Serum-free versions of such culture media are alsoavailable. Cell culture media may be supplemented with additional orincreased concentrations of components such as amino acids, salts,sugars, vitamins, hormones, growth factors, buffers, antibiotics,lipids, trace elements and the like, depending on the requirements ofthe cells to be cultured and/or the desired cell culture parameters.

Cell culture media may be serum-free, protein-free, and/or peptone-free.“Serum-free” applies to a cell culture medium that does not containanimal sera, such as fetal bovine serum. “Protein-free” applies to cellculture media free from exogenously added protein, such as transferrin,protein growth factors IGF-1, or insulin. Protein-free media may or maynot contain peptones. “Peptone-free” applies to cell culture media whichcontains no exogenous protein hydrolysates such as animal and/or plantprotein hydrolysates. Cell culture broth, or like terminology, refers tothe cell culture media that contains, among other things, viable andnon-viable mammalian cells, cell metabolites and cellular debris such asnucleic acids, proteins and liposomes.

For upregulating the high mannose glycoform content of a recombinantantibody, the cell culture media of the present invention containsmannose, wherein the mannose to total hexose ratio in the cell culturemedia is greater than 0 but less than 1.0. The ratio may be 0.1, 0.2,0.3, 0.5, 0.6, 0.7, 0.8, 0.9, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40,0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.5, 0.90, 0.91, 0.92,0.93, 0.94, 0.95, 0.96, 0.97, 0.98 or 0.99 or any value in between. Theconcentration of mannose in the cell culture media is preferably atleast 3 g/l, at least 6 g/L, at least 9 g/L, or at least 11 g/L.Preferably the concentration of mannose is greater than 11 g/L.Preferably the concentration of mannose is 11.01, 11.05, 11.10, 11.15,11.20, 11.25, 11.30, 11.35, 11.40, 11.45, 11.50, 11.55, 11.60, 11.65,11.70, 11.75, 11.80, 11.85, 11.90, 11.95 g/L or any value in between. Ina preferred embodiment, the amount of mannose is 11.35 g/L. Preferablythe cell culture media also contains glucose at such a concentrationthat the mannose to hexose ratio is greater than 0 but less than 1.0. Inone embodiment the amount of glucose is at least 0.75 g/L to 12 g/L. Ina preferred embodiment the amount of glucose is 0.75 g/L.

Cell cultures can also be supplemented with concentrated feed mediumcontaining components, such as nutrients and amino acids, which areconsumed during the course of the production phase of the cell culture,see for example WIPO Publication No WO2012/145682. Concentrated feedmedium may be based on just about any cell culture media formulation.Such a concentrated feed medium can contain anywhere from a single ornearly almost all of the components of the cell culture medium at, forexample, about 5×, 6×, 7×, 8×, 9×, 10×, 12×, 14×, 16×, 20×, 30×, 50×,100×, 200×, 400×, 600×, 800×, or even about 1000× of their normalamount.

By “cell culture” or “culture” is meant the growth and propagation ofcells outside of a multicellular organism or tissue. Suitable cultureconditions for mammalian cells are known in the art. See e.g. Animalcell culture: A Practical Approach, D. Rickwood, ed., Oxford UniversityPress, New York (1992). Mammalian cells may be cultured in suspension orwhile attached to a solid substrate. Fluidized bed bioreactors, hollowfiber bioreactors, roller bottles, shake flasks, or stirred tankbioreactors, with or without microcarriers, can be used.

The mammalian cell culture is grown in a bioreactor. In one embodiment500 L to 20000 L bioreactors are used. In a preferred embodiment, 1000 Lto 2000 L bioreactors are used.

The bioreactor may be inoculated with at least 0.5×10⁶ and up to andbeyond 3.0×10⁶ viable cells/mL in a serum-free culture medium. In apreferred embodiment the inoculation is 1.0×10⁶ viable cells/mL.

Once inoculated into the production bioreactor the mammalian cellsundergo an exponential growth phase. The growth phase can be maintainedas a batch culture, using a fed-batch process with bolus feeds of a feedmedium, as a perfusion culture, or any combination. For fed batchmethods, supplemental bolus feeds typically begin shortly after thecells are inoculated into the bioreactor, at a time when it isanticipated, estimated or determined that the cell culture needsfeeding. For example, supplemental feeds can begin on or about day 3 or4 of the culture or a day or two earlier or later. The culture mayreceive two, three, or more bolus feeds during the growth phase. Forcultures maintained by perfusion, perfusion feeds can begin at any time,for example, perfusion feeds can begin on or about day 3 or 4 of theculture or a day or two earlier or later.

When the cells enter the stationary or production phase, or the cellculture has achieved a desired viable cell density and/or cell titer,the fed batch bolus feeds can be discontinued and perfusion feedsstarted. Perfusion culture is one in which the cell culture receivesfresh perfusion feed medium while simultaneously removing spent medium.Perfusion can be continuous, step-wise, intermittent, or a combinationof any or all of any of these. Perfusion rates can be less than aworking volume to many working volumes per day. Preferably the cells areretained in the culture and the spent medium that is removed issubstantially free of cells or has significantly fewer cells than theculture. Perfusion can be accomplished by a number of means includingcentrifugation, sedimentation, or filtration, See e.g. Voisard et al.,(2003), Biotechnology and Bioengineering 82:751-65. A preferredfiltration method is alternating tangential flow filtration. Alternatingtangential flow is maintained by pumping medium through hollow-fiberfilter modules. See e.g. U.S. Pat. No. 6,544,424. The hollow-fibermodules can be microfilters or ultrafilters.

When the fed-batch culture reaches a predetermined trigger point, suchas desired cell viability, cell density, percent packed cell volume,titer, packed cell volume adjusted titer, age or the like, a switchbetween fed-batch and perfusion can take place. For example, this switchcan take place on or about day 7 of the culture, but may take place aday or two earlier or later. The perfusion feed formulation may containglucose at a concentration of up to 14 g/L or more. In one embodiment,the perfusion medium contains 12 g/L glucose.

Upregulation of glycoform content can begin at any stage at or after thecells are inoculated into the production bioreactor, during the growthphase, at the start of the production phase, during the productionphase. Bolus feeds and/or perfusion media may contain concentrations ofmannose such as to give a mannose to total hexose ratio of greater than0 but less than 1.0.

When the perfusion culture reaches a predetermined trigger point, suchas desired cell viability, cell density, percent packed cell volume,titer, packed cell volume adjusted titer, age or the like, mannose maybe added to the media such as to give a mannose to total hexose ratio ofgreater than 0 but less than 1.0. For example, this shift may beinitiated on day 11 of the culture, but may take place a day or twoearlier or later. At that time the cell culture is perfused with cellculture medium containing mannose such that the mannose to total hexoseratio is greater than 0 but less than 1.0. The ratio may be 0.1, 0.2,0.3, 0.5, 0.6, 0.7, 0.8, 0.9, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40,0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.5, 0.90, 0.91, 0.92,0.93, 0.94, 0.95, 0.96, 0.97, 0.98 or 0.99 or any value in between. Theconcentration of mannose in the cell culture media is preferably atleast 3 g/l, at least 6 g/L, at least 9 g/L, or at least 11 g/L.Preferably the concentration of mannose is greater than 11 g/L.Preferably the concentration of mannose is 11.01, 11.05, 11.10, 11.15,11.20, 11.25, 11.30, 11.35, 11.40, 11.45, 11.50, 11.55, 11.60, 11.65,11.70, 11.75, 11.80, 11.85, 11.90, 11.95 g/L or any value in between. Ina preferred embodiment, the amount of mannose is 11.35 g/L. Preferablythe cell culture media also contains glucose at such a concentrationthat the mannose to hexose ratio is greater than 0 but less than 1.0. Inanother embodiment the amount of glucose is at least 0.75 g/L to 12 g/L.In one embodiment, the amount of glucose is 0.75 g/L.

The mannose to total hexose ratio in the cell culture is determined andmaintained by monitoring the concentration of mannose and hexose sugarsin the cell culture media added to the bioreactor, and adjusting themannose and hexose sugar concentration in the medium formulation tomaintain a ratio of greater than 0 but less than 1.0. Samples may alsobe taken from the bioreactor or the spent media to determine the mannoseto total hexose ratio.

The cell culture can be continuously maintained or harvested. The cellculture can be restored to a state where the mannose to total hexoseratio is greater than 1.0 via supplements to the cell culture media or areformulation of the feed media and the entire process begun again as isdesired.

The cell culture could also be maintained in a perfusion culture systemfor both the growth and production phases. Once inoculated into theproduction bioreactor the mammalian cells undergo an exponential growthphase. If desired, the cell culture may be supplemented with mannosesuch as to maintain a mannose to total hexose ratio of greater than 0but less than 1.0. The culture is maintained until a desired triggerpoint is achieved, for example, desired viable cell density, cellviability, percent packed cell volume, titer, packed cell adjustedvolume titer, age or the like, that signals the start of a productionphase. At that time or at any time during the production phase, the cellculture may be perfused with a cell culture medium supplemented withmannose such that the mannose to total hexose ratio is greater than 0but less than 1.0. The ratio may be 0.1, 0.2, 0.3, 0.5, 0.6, 0.7, 0.8,0.9, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60,0.65, 0.70, 0.75, 0.80, 0.5, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96,0.97, 0.98 or 0.99 or any value in between. The concentration of mannosein the cell culture media is preferably at least 3 g/l, at least 6 g/L,at least 9 g/L, or at least 11 g/L or more. Preferably the concentrationof mannose is greater than 11 g/L. Preferably the concentration ofmannose is 11.01, 11.05, 11.10, 11.15, 11.20, 11.25, 11.30, 11.35,11.40, 11.45, 11.50, 11.55, 11.60, 11.65, 11.70, 11.75, 11.80, 11.85,11.90, 11.95 g/L or any value in between. In a preferred embodiment, theamount of mannose is 11.35 g/L. Preferably the cell culture media alsocontains glucose at such a concentration that the mannose to hexoseratio is greater than 0 but less than 1.0. In another embodiment theamount of glucose is at least 0.75 g/L to 12 g/L. In one embodiment, theamount of glucose is 0.75 g/L.

Viable cell density may be a signal for transition to the productionphase or trigger use of a cell culture media having a mannose to totalhexose ratio of greater than 0 but less than 1.0 to the cell culturemedia. It may also be desirable to maintain a certain range or level ofviable cell density during the production phase or while using a cellculture media having a mannose to total hexose ratio is greater than 0bur less than 1.0. In one embodiment the viable cell density is 10×10⁶viable cells/mL to at least about 60×10⁶ viable cells/mL. In anotherembodiment the viable cell density is 10×10⁶ viable cells/mL to 50×10⁶viable cells/mL. In another embodiment the viable cell density is 10×10⁶viable cells/mL to 40×10⁶ viable cells/mL. In a preferred embodiment theviable cell density is 10×10⁶ viable cells/mL to 30×10⁶ viable cells/mL.In another preferred embodiment the viable cell density is 10×10⁶ viablecells/mL to 20×10⁶ viable cells/mL. In another preferred embodiment theviable cell density is 20×10⁶ viable cells/mL to 30×10⁶ viable cells/mL.In yet another preferred embodiment the viable cell density is 20×10⁶viable cells/mL to 25×10⁶ viable cells/mL. In an even more preferredembodiment the viable cell density is at least about 20×10⁶ viablecells/mL.

The percent packed cell volume (% PCV) may also be used as a signal fortransition to the production phase or trigger use of a cell culturemedia having a mannose to total hexose ratio of greater than 0 but lessthan 1.0 to the cell culture media. The cell culture may also bemaintained at a desired packed cell volume during the production phaseor while using a cell culture media having a mannose to total hexoseratio of 0 to 1.0. In one embodiment the packed cell volume is equal toor less than 30%. In a preferred embodiment the packed cell volume is atleast about 15-30%. In a preferred embodiment the packed cell volume isat least about 20-25%. In another preferred embodiment the packed cellvolume is equal to or less than 25%. In another preferred embodiment thepacked cell volume is equal to or less than 15%. In another preferredembodiment the packed cell volume is equal to or less than 20%. In yetanother preferred embodiment the packed cell volume is equal to or lessthan 15%.

A perfusion cell culture medium having a reduced concentration ofasparagine can be used to arrest cell growth while maintainingproductivity and viability during the production phase or in a cellculture media having a mannose to total hexose ratio of greater than 0to less than 1.0. In a preferred embodiment the concentration ofasparagine is at least about 0 mM to at least about 5 mM asparagine, seeWIPO Publication No. WO 2013/006479.

As used herein, “perfusion flow rate” is the amount of media that ispassed through (added and removed) from a bioreactor, typicallyexpressed as some portion or multiple of the working volume, in a giventime. “Working volume” refers to the amount of bioreactor volume usedfor cell culture. In one embodiment the perfusion flow rate is oneworking volume or less per day. Perfusion feed medium can be formulatedto maximize perfusion nutrient concentration to minimize perfusion rate.

As used herein, “cell density” refers to the number of cells in a givenvolume of culture medium. “Viable cell density” refers to the number oflive cells in a given volume of culture medium, as determined bystandard viability assays (such as trypan blue dye exclusion method).

As used herein, “packed cell volume” (PCV), also referred to as “percentpacked cell volume” (% PCV), is the ratio of the volume occupied by thecells, to the total volume of cell culture, expressed as a percentage(see Stettler, wt al., (2006) Biotechnol Bioeng. Dec 20:95(6):1228-33).Packed cell volume is a function of cell density and cell diameter;increases in packed cell volume could arise from increases in eithercell density or cell diameter or both. Packed cell volume is a measureof the solid content in the cell culture. Solids are removed duringharvest and downstream purification. More solids mean more effort toseparate the solid material from the desired product during harvest anddownstream purification steps. Also, the desired product can becometrapped in the solids and lost during the harvest process, resulting ina decreased product yield. Since host cells vary in size and cellcultures also contain dead and dying cells and other cellular debris,packed cell volume is a more accurate way to describe the solid contentwithin a cell culture than cell density or viable cell density.

The method according to the present invention may be used to improve theproduction of recombinant proteins in a multiple phase culture process.In a multiple stage process, cells are cultured in two or more distinctphases. For example cells may be cultured first in one or more growthphases, under environmental conditions that maximize cell proliferationand viability, then transferred to a production phase, under conditionsthat maximize protein production. In a commercial process for productionof a protein by mammalian cells, there are commonly multiple, forexample, at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more growthphases that occur in different culture vessels preceding a finalproduction culture. The growth and production phases may be preceded by,or separated by, one or more transition phases. In multiple phaseprocesses, the method according to the present invention can be employedat least during the growth and production phase of the final productionphase of a commercial cell culture, although it may also be employed inany of the preceding growth, transition and/or production phases. Aproduction phase can be conducted at large scale. A large scale processcan be conducted in a volume of at least about 100, 500, 1000, 2000,3000, 5000, 7000, 8000, 10,000, 15,000, or 20,000 liters or more. In apreferred embodiment production is conducted in 500 L, 1000 L and/or2000 L bioreactors. A growth phase may occur at a higher temperaturethan a production phase. For example, a growth phase may occur at afirst temperature from about 35° C. to about 38° C., and a productionphase may occur at a second temperature from about 29° C. to about 37°C., optionally from about 30° C. to about 36° C. or from about 30° C. toabout 34° C. In addition, chemical inducers of protein production, suchas, for example, caffeine, butyrate, and/or hexamethylene bisacetamide(HMBA), may be added at the same time as, before, and/or after atemperature shift. If inducers are added after a temperature shift, theycan be added from one hour to five days after the temperature shift,optionally from one to two days after the temperature shift. The cellcultures can be maintained for days or even weeks while the cellsproduce the desired protein(s).

Typically the cell cultures that precede the final production culture(N−x to N−1) are used to generate the seed cells that will be used toinoculate the production bioreactor, the N−1 culture. The seed celldensity can have a positive impact on the level of recombinant proteinproduced. Product levels tend to increase with increasing seed density.Improvement in titer is tied not only to higher seed density, but islikely to be influenced by the metabolic and cell cycle state of thecells that are placed into production.

Seed cells can be produced by any culture method. A preferred method isa perfusion culture using alternating tangential flow filtration. An N−1bioreactor can be run using alternating tangential flow filtration toprovide cells at high density to inoculate a production bioreactor. TheN−1 stage may be used to grow cells to densities of >90×10⁶ cells/mL.The N−1 bioreactor can be used to generate bolus seed cultures or can beused as a rolling seed stock culture that could be maintained to seedmultiple production bioreactors at high seed cell density. The durationof the growth stage of production can range from 7 to 14 days and can bedesigned so as to maintain cells in exponential growth prior toinoculation of the production bioreactor. Perfusion rates, mediumformulation and timing are optimized to grow cells and deliver them tothe production bioreactor in a state that is most conducive tooptimizing their production. Seed cell densities of >15×10⁶ cells/mL canbe achieved for seeding production bioreactors.

The cell lines (also referred to as “host cells”) used in the inventionare genetically engineered to express a polypeptide of commercial orscientific interest. Cell lines are typically derived from a lineagearising from a primary culture that can be maintained in culture for anunlimited time. Genetically engineering the cell line involvestransfecting, transforming or transducing the cells with a recombinantpolynucleotide molecule, and/or otherwise altering (e.g., by homologousrecombination and gene activation or fusion of a recombinant cell with anon-recombinant cell) so as to cause the host cell to express a desiredrecombinant polypeptide. Methods and vectors for genetically engineeringcells and/or cell lines to express a polypeptide of interest are wellknown to those of skill in the art; for example, various techniques areillustrated in Current Protocols in Molecular Biology, Ausubel et al.,eds. (Wiley & Sons, New York, 1988, and quarterly updates); Sambrook etal., Molecular Cloning: A Laboratory Manual (Cold Spring LaboratoryPress, 1989); Kaufman, R. J., Large Scale Mammalian Cell Culture, 1990,pp. 15-69.

Animal cell lines are derived from cells whose progenitors were derivedfrom a multi-cellular animal. One type of animal cell line is amammalian cell line. A wide variety of mammalian cell lines suitable forgrowth in culture are available from the American Type CultureCollection (Manassas, Va.) and commercial vendors. Examples of celllines commonly used in the industry include VERO, BHK, HeLa, CV1(including Cos), MDCK, 293, 3T3, myeloma cell lines (e.g., NSO, NS1),PC12, WI38 cells, and Chinese hamster ovary (CHO) cells. CHO cells arewidely used for the production of complex recombinant proteins, e.g.cytokines, clotting factors, and antibodies (Brasel et al. (1996), Blood88:2004-2012; Kaufman et al. (1988), J Biol Chem 263:6352-6362; McKinnonet al. (1991), J Mol Endocrinol 6:231-239; Wood et al. (1990), J.Immunol. 145:3011-3016). The dihydrofolate reductase (DHFR)-deficientmutant cell lines (Urlaub et al. (1980), Proc Natl Acad Sci USA 77:4216-4220), DXB11 and DG-44, are desirable CHO host cell lines becausethe efficient DHFR selectable and amplifiable gene expression systemallows high level recombinant protein expression in these cells (KaufmanR. J. (1990), Meth Enzymol 185:537-566). In addition, these cells areeasy to manipulate as adherent or suspension cultures and exhibitrelatively good genetic stability. CHO cells and proteins recombinantlyexpressed in them have been extensively characterized and have beenapproved for use in clinical commercial manufacturing by regulatoryagencies.

The methods of the invention can be used to culture cells that expressrecombinant proteins of interest. The expressed recombinant proteins maybe secreted into the culture medium from which they can be recoveredand/or collected. In addition, the proteins can be purified, orpartially purified, from such culture or component (e.g., from culturemedium) using known processes and products available from commercialvendors. The purified proteins can then be “formulated”, meaning bufferexchanged, sterilized, bulk-packaged, and/or packaged for a final user.Suitable formulations for pharmaceutical compositions include thosedescribed in Remington's Pharmaceutical Sciences, 18th ed. 1995, MackPublishing Company, Easton, Pa.

As used herein “peptide,” “polypeptide” and “protein” are usedinterchangeably throughout and refer to a molecule comprising two ormore amino acid residues joined to each other by peptide bonds.Polypeptides can be of scientific or commercial interest, includingprotein-based drugs. Polypeptides include, among other things,antibodies, fusion proteins, and cytokines. Peptides, polypeptides andproteins can be produced by recombinant animal cell lines using cellculture methods and may be referred to as “recombinant peptide”,“recombinant polypeptide” and “recombinant protein”. The expressedprotein(s) may be produced intracellularly or secreted into the culturemedium from which it can be recovered and/or collected.

Peptides, polypeptides and proteins are also inclusive of modificationsincluding, but not limited to, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation. “Glycoprotein” refers to peptides, polypeptidesand proteins, having at least one oligosaccharide side chain includingmannose residues. Glycoproteins may be homologous to the host cell, ormay be heterologous, i.e., foreign, to the host cell being utilized,such as, for example, a human glycoprotein produced by a Chinese hamsterovary (CHO) host-cell.

Examples of polypeptides that can be produced with the methods of theinvention include proteins comprising amino acid sequences identical toor substantially similar to all or part of one of the followingproteins: tumor necrosis factor (TNF), flt3 ligand (WO 94/28391),erythropoeitin, thrombopoeitin, calcitonin, IL-2, angiopoietin-2(Maisonpierre et al. (1997), Science 277(5322): 55-60), ligand forreceptor activator of NF-kappa B (RANKL, WO 01/36637), tumor necrosisfactor (TNF)-related apoptosis-inducing ligand (TRAIL, WO 97/01633),thymic stroma-derived lymphopoietin, granulocyte colony stimulatingfactor, granulocyte-macrophage colony stimulating factor (GM-CSF,Australian Patent No. 588819), mast cell growth factor, stem cell growthfactor (U.S. Pat. No. 6,204,363), epidermal growth factor, keratinocytegrowth factor, megakaryote growth and development factor, RANTES, humanfibrinogen-like 2 protein (FGL2; NCBI accession no. NM_00682; Rüegg andPytela (1995), Gene 160:257-62) growth hormone, insulin, insulinotropin,insulin-like growth factors, parathyroid hormone, interferons includingα-interferons, γ-interferon, and consensus interferons (U.S. Pat. Nos.4,695,623 and 4,897,471), nerve growth factor, brain-derivedneurotrophic factor, synaptotagmin-like proteins (SLP 1-5),neurotrophin-3, glucagon, interleukins, colony stimulating factors,lymphotoxin-β, leukemia inhibitory factor, and oncostatin-M.Descriptions of other glycoproteins may be found in, for example, HumanCytokines: Handbook for Basic and Clinical Research, all volumes(Aggarwal and Gutterman, eds. Blackwell Sciences, Cambridge, Mass.,1998); Growth Factors: A Practical Approach (McKay and Leigh, eds.,Oxford University Press Inc., New York, 1993); and The CytokineHandbook, Vols. 1 and 2 (Thompson and Lotze eds., Academic Press, SanDiego, Calif., 2003).

Additionally the methods of the invention would be useful to produceproteins comprising all or part of the amino acid sequence of a receptorfor any of the above-mentioned proteins, an antagonist to such areceptor or any of the above-mentioned proteins, and/or proteinssubstantially similar to such receptors or antagonists. These receptorsand antagonists include: both forms of tumor necrosis factor receptor(TNFR, referred to as p55 and p75, U.S. Pat. Nos. 5,395,760 and5,610,279), Interleukin-1 (IL-1) receptors (types I and II; EP PatentNo. 0460846, U.S. Pat. Nos. 4,968,607, and 5,767,064,), IL-1 receptorantagonists (U.S. Pat. No. 6,337,072), IL-1 antagonists or inhibitors(U.S. Pat. Nos. 5,981,713, 6,096,728, and 5,075,222) IL-2 receptors,IL-4 receptors (EP Patent No. 0 367 566 and U.S. Pat. No. 5,856,296),IL-15 receptors, IL-17 receptors, IL-18 receptors, Fc receptors,granulocyte-macrophage colony stimulating factor receptor, granulocytecolony stimulating factor receptor, receptors for oncostatin-M andleukemia inhibitory factor, receptor activator of NF-kappa B (RANK, WO01/36637 and U.S. Pat. No. 6,271,349), osteoprotegerin (U.S. Pat. No.6,015,938), receptors for TRAIL (including TRAIL receptors 1, 2, 3, and4), and receptors that comprise death domains, such as Fas orApoptosis-Inducing Receptor (AIR).

Other proteins that can be produced using the invention include proteinscomprising all or part of the amino acid sequences of differentiationantigens (referred to as CD proteins) or their ligands or proteinssubstantially similar to either of these. Such antigens are disclosed inLeukocyte Typing VI (Proceedings of the VIth International Workshop andConference, Kishimoto, Kikutani et al., eds., Kobe, Japan, 1996).Similar CD proteins are disclosed in subsequent workshops. Examples ofsuch antigens include CD22, CD27, CD30, CD39, CD40, and ligands thereto(CD27 ligand, CD30 ligand, etc.). Several of the CD antigens are membersof the TNF receptor family, which also includes 41BB and OX40. Theligands are often members of the TNF family, as are 41BB ligand and OX40ligand.

Enzymatically active proteins or their ligands can also be produced bythe invention. Examples include proteins comprising all or part of oneof the following proteins or their ligands or a protein substantiallysimilar to one of these: a disintegrin and metalloproteinase domainfamily members including TNF-alpha Converting Enzyme, various kinases,glucocerebrosidase, superoxide dismutase, tissue plasminogen activator,Factor VIII, Factor IX, apolipoprotein E, apolipoprotein A-I, globins,an IL-2 antagonist, alpha-1 antitrypsin, ligands for any of theabove-mentioned enzymes, and numerous other enzymes and their ligands.

The term “antibody” includes reference to immunoglobulins of any isotypeor subclass or to an antigen-binding region thereof that competes withthe intact antibody for specific binding, unless otherwise specified,including human, humanized, chimeric, multi-specific, monoclonal,polyclonal, and oligomers or antigen binding fragments thereof. Alsoincluded are proteins having an antigen binding fragment or region suchas Fab, Fab′, F(ab′)₂, Fv, diabodies, Fd, dAb, maxibodies, single chainantibody molecules, complementarity determining region (CDR) fragments,scFv, diabodies, triabodies, tetrabodies and polypeptides that containat least a portion of an immunoglobulin that is sufficient to conferspecific antigen binding to a target polypeptide. The term “antibody” isinclusive of, but not limited to, those that are prepared, expressed,created or isolated by recombinant means, such as antibodies isolatedfrom a host cell transfected to express the antibody.

Examples of antibodies include, but are not limited to, those thatrecognize any one or a combination of proteins including, but notlimited to, the above-mentioned proteins and/or the following antigens:CD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD20, CD22, CD23, CD25, CD33,CD40, CD44, CD52, CD80 (B7.1), CD86 (B7.2), CD147, IL-1α, IL-1β, IL-2,IL-3, IL-7, IL-4, IL-5, IL-8, IL-10, IL-2 receptor, IL-4 receptor, IL-6receptor, IL-13 receptor, IL-18 receptor subunits, FGL2, PDGF-β andanalogs thereof (see U.S. Pat. Nos. 5,272,064 and 5,149,792), VEGF, TGF,TGF-β2, TGF-β1, EGF receptor (see U.S. Pat. No. 6,235,883) VEGFreceptor, hepatocyte growth factor, osteoprotegerin ligand, interferongamma, B lymphocyte stimulator (BlyS, also known as BAFF, THANK, TALL-1,and zTNF4; see Do and Chen-Kiang (2002), Cytokine Growth Factor Rev.13(1): 19-25), C5 complement, IgE, tumor antigen CA125, tumor antigenMUC1, PEM antigen, LCG (which is a gene product that is expressed inassociation with lung cancer), HER-2, HER-3, a tumor-associatedglycoprotein TAG-72, the SK-1 antigen, tumor-associated epitopes thatare present in elevated levels in the sera of patients with colon and/orpancreatic cancer, cancer-associated epitopes or proteins expressed onbreast, colon, squamous cell, prostate, pancreatic, lung, and/or kidneycancer cells and/or on melanoma, glioma, or neuroblastoma cells, thenecrotic core of a tumor, integrin alpha 4 beta 7, the integrin VLA-4,B2 integrins, TRAIL receptors 1, 2, 3, and 4, RANK, RANK ligand, TNF-α,the adhesion molecule VAP-1, epithelial cell adhesion molecule (EpCAM),intercellular adhesion molecule-3 (ICAM-3), leukointegrin adhesin, theplatelet glycoprotein gp IIb/IIIa, cardiac myosin heavy chain,parathyroid hormone, rNAPc2 (which is an inhibitor of factor VIIa-tissuefactor), MHC I, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP),tumor necrosis factor (TNF), CTLA-4 (which is a cytotoxic Tlymphocyte-associated antigen), Fc-γ-1 receptor, HLA-DR 10 beta, HLA-DRantigen, sclerostin, L-selectin, Respiratory Syncitial Virus, humanimmunodeficiency virus (HIV), hepatitis B virus (HBV), Streptococcusmutans, and Staphylococcus aureus. Specific examples of known antibodieswhich can be produced using the methods of the invention include but arenot limited to adalimumab, bevacizumab, infliximab, abciximab,alemtuzumab, bapineuzumab, basiliximab, belimumab, briakinumab,canakinumab, certolizumab pegol, cetuximab, conatumumab, denosumab,eculizumab, gemtuzumab ozogamicin, golimumab, ibritumomab tiuxetan,labetuzumab, mapatumumab, matuzumab, mepolizumab, motavizumab,muromonab-CD3, natalizumab, nimotuzumab, ofatumumab, omalizumab,oregovomab, palivizumab, panitumumab, pemtumomab, pertuzumab,ranibizumab, rituximab, rovelizumab, tocilizumab, tositumomab,trastuzumab, ustekinumab, vedolizomab, zalutumumab, and zanolimumab.

The invention can be used to produce recombinant fusion proteinscomprising, for example, any of the above-mentioned proteins. Forexample, recombinant fusion proteins comprising one of theabove-mentioned proteins plus a multimerization domain, such as aleucine zipper, a coiled coil, an Fc portion of an immunoglobulin, or asubstantially similar protein, can be produced using the methods of theinvention. See e.g. WO94/10308; Lovejoy et al. (1993), Science259:1288-1293; Harbury et al. (1993), Science 262:1401-05; Harbury etal. (1994), Nature 371:80-83; H{dot over (a)}kansson et al. (1999),Structure 7:255-64. Specifically included among such recombinant fusionproteins are proteins in which a portion of a receptor is fused to an Fcportion of an antibody such as etanercept (a p75 TNFR:Fc), andbelatacept (CTLA4:Fc). In another embodiment are antibody-drugconjugates.

While the terminology used in this application is standard within theart, definitions of certain terms are provided herein to assure clarityand definiteness to the meaning of the claims. Units, prefixes, andsymbols may be denoted in their SI accepted form. Numeric ranges recitedherein are inclusive of the numbers defining the range and include andare supportive of each integer within the defined range. Unlessotherwise noted, the terms “a” or “an” are to be construed as meaning“at least one of”. The section headings used herein are fororganizational purposes only and are not to be construed as limiting thesubject matter described. The methods and techniques described hereinare generally performed according to conventional methods well known inthe art and as described in various general and more specific referencesthat are cited and discussed throughout the present specification unlessotherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: ALaboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2001) and Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing Associates (1992), and Harlow andLane Antibodies: A Laboratory Manual Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1990). All documents, or portions ofdocuments, cited in this application, including but not limited topatents, patent applications, articles, books, and treatises, are herebyexpressly incorporated by reference.

The present invention is not to be limited in scope by the specificembodiments described herein that are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

The following examples demonstrate embodiments and aspects of thedisclosed methods and are not intended to be limiting.

EXAMPLES Example 1

A small scale mock perfusion model was used to evaluate the effects ofdifferent carbon sources and their concentrations on high mannoseglycoform modulation, cell growth, antibody production. A mock perfusionassay was performed in a 24 deep-well plate (Axygen, Union City,Calif.). Serum free, defined cell culture media were prepared containing12 g/L mannose, galactose, fructose, maltose or glucose (sugars obtainedfrom Sigma-Aldrich, St. Louis, Mo.) as the sole carbon source. Theglucose containing media acted as the control. Briefly, each of the cellculture media were tested by using a CHO cell line expressing an IgG2recombinant antibody (Cell line A) seeded at a targeted density rangingfrom 20 to 30×10⁶ cells/mL in 3 mL of cell culture media per well. Thecells were cultivated at 36° C., 5% CO₂, 85% relative humidity andshaken at 225 rpm in a 50-mm orbital diameter Kuhner incubator (KuhnerAG, Basel, Switzerland) for 3 or 4 days. Every 24 hours, cells werecentrifuged at 200×g for 5 minutes (Beckman Coulter, Brea, Calif.) tocollect the spent media and each well was then replenished with 3 mLfresh media. The collected spent media was analyzed for titer,viability, viable cell density and percent high mannose glycoformcontent (% HM). The cells were harvested on day 3 or 4 and cell countsand viability were measured. All experimental conditions were performedin duplicate.

Viable cell density and viability were determined using a Cedex cellcounter (Roche Innovative, Beilefed, Germany). Metabolite information(glucose, lactate, ammonia, glutamine, glutamate) was obtained usingNovaBioprofile Flex (Nova Biomedical, Waltham, Mass.). Antibodyconcentration in the spent media was determined using a Affinity ProteinA Ultra Performance liquid chromatography (UPLC) (Waters Corporation,Milford, Mass.) equipped with a 50 mm×4.6 mm i.d. POROS A/20 protein Acolumn (Life Technologies, Carlsbad, Calif.). After the sample wasinjected, the column was washed with Phosphate-Buffered Saline (PBS)pH=7.1 to remove CHO host cell proteins. Bound antibodies were theneluted in acidic PBS buffer (pH=1.9) and detected by UV absorbance at280 nm to quantify antibody concentration. Packed cell volume wasmeasured by spinning cells at 1462×g for 17 minutes. The adjusted titerwas then calculated.

A high throughput microchip capillary electrophoresis method was used todetermine the high mannose glycoform content on the recombinantantibodies using samples taken directly from the spent media. Sampleswere digested with endoglycosidase H (Endo H) for 2 hours at 37° C.After digestion, antibodies were denatured at 70° C. for 10 minutes andinjected in to a LabChip GXII (Caliper Life Sciences, Hopkinton, Mass.)to quantify the amount of non-glycosylated heavy chain (NGHC). Samplesnot digested by Endo H were used as the controls. The % of NGHC from thecontrol was subtracted by the % NGHC of test samples to yield thepercent high mannose glycoform content (HM %) value.

When galactose, fructose and maltose were metabolized, cell growth(viable cell density) was significantly inhibited as shown in FIG. 1A.Additionally, the viability and antibody titer were also negativelyimpacted (FIGS. 1B and 1C). After analysis of antibody qualityattributes, it was found that the high mannose glycoform content was 59%in the galactose, fructose and maltose cultures, about five-fold higherthan that of the glucose control (12%) (FIG. 1D). Interestingly, thosecell cultures with mannose as the sole carbon source yielded growth,viability and antibody titer comparable with the glucose control (FIG.1A to 1C). In the mannose cultures, viable cell density (VCD) continuedto increase and reached 52×10⁶ cells/mL with viability at 94% at day 3,and an antibody titer of 3.4 g/L. However, HM was 35% (FIG. 1D). Thisindicates that presence of mannose in the media affects glycanprocessing while maintaining growth and protein production levelsequivalent to cell cultures grown in media supplemented with glucoseonly. Mannose in the cell culture media maintained cell growth andprotein production equivalent to cultures grown in glucose media, whileincreasing upregulating high mannose glycoform content.

Based on the above results it was hypothesized that mannoseconcentration in the media could influence the high mannose glycoformcontent of the recombinant antibody being expressed. CHO cells mayuptake mannose at a rate similar to glucose, so varying the ratio ofmannose to glucose may yield different levels of high mannose glycoformcontent while having no negative impact on cell growth and antibodyproduction. To test this hypothesis, serum free, defined cell culturemedia were prepared with glucose and mannose concentrations as shown inTable 1.

TABLE 1 Experimental design of using different glucose and mannoseconcentration in the media to study high mannose modulation effect.Mannose/ Total Hexose Glucose Mannose (M/H) Media (g/L) (g/L) ratioG12M0 (control) 12 0 0 G9M3 9 3 0.25 G6M6 6 6 0.5 G3M9 3 9 0.75 G0M120.75 11.25 0.94

When the amount of glucose in the cell culture media was reduced andreplaced with 3, 6, 9 and 11.25 g/L of mannose, the high mannoseglycoform content increased from 12% to 16, 21, 27 and 33%, respectively(FIG. 2A). The viable cell density, viability and antibody titer werenot affected compared to the 12 g/L glucose control (FIGS. 2 B, C andD). The results show that varying the ratio of mannose to glucose in thecell culture media could modulate the high mannose glycoform content (%HM) without alternating growth and productivity and that a linearcorrelation existed between percent high mannose glycoform content onthe recombinant antibody and the mannose concentration provided in thecell culture media.

To test whether the modulation of the high mannose glycoform content wasapplicable across different antibody expressing cell lines, four otherCHO cell lines (B, C, D and E) expressing IgG1 or IgG2 antibodies, werecultivated in the media containing the same mannose/glucoseconcentrations shown in Table 1. After harvest, samples were analyzed,and % HM value was plotted against the different mannose/total hexose(M/H) ratios of the media. As indicated in FIGS. 3A-3E, these four celllines were all shown to have elevated % HM at high M/H ratio as seeabove for Cell line A. This result is striking especially consideringthat these cell lines have inherently different % HM for theirrespective antibodies. For example, cell line C produced an antibodywith 2% HM in the control media with glucose only (M/H ratio 0.00) butthe % HM increased to 4.4% at a M/H ratio of 0.94 (mannose at 11.25g/L), FIG. 3C. On the other hand, Cell line B which would be considereda high % HM cell line, having a HM of 35% in the control media (M/Hratio 0.00), increased to 69% at the M/H ratio of 0.94 (FIG. 3B). Astrong linear correlation between HM % and the M/H ratio was seen in allof the cell lines tested. This indicated that % HM of the producedantibodies could be controlled by specific M/H ratio in the cell culturemedia making the % HM of the produced antibodies predictable.

Example 2

To study the effect of different glucose/mannose ratios on HM % and toverify that the HM modulation effect is scalable, bioreactor runs usingCell line A were conducted. Three 3 L bench top bioreactors (ApplikonBiotechnology, Forester City, Calif.) were equipped with ATF 2 systems(Refine Technology, Pine Brook, N.J.) and 30 kDa cut-off hallow fibercartridges (GE Healthcare Biosciences, Pittsburgh, Pa.) for mediaperfusion throughout the culture.

CHO Cell line A was seeded at 1.4×10⁶ cells/mL in the bioreactorscontaining 1.5 L of serum free, defined growth media containing 6.25 g/Lglucose as the only carbon source. An automated bioprocessing softwarewas used for data acquisition and parameter control. Starting on day 3,the bioreactors were perfused with serum free, defined cell culturemedia containing different concentrations of glucose and mannose as theonly carbon sources, see Table 2.

TABLE 2 Glucose and mannose concentration in perfusion cell culturemedia Mannose/ Total Glucose Mannose Hexose (g/L) (g/L) (M/H) ratio 12 00 (control) 6 6 0.5 0.75 11.25 0.94

During the culture process the cells were initially grown at 36° C. withagitation of 150 rpm and an air flow rate of 100 cm³/min, and shifted to33° C. once the cell density reached 35 to 40×10⁶ cells/mL. Thedissolved oxygen level was maintained at 60% of air saturation andsupplemented with pure oxygen when needed. The pH of the culture mediawas maintained at 7.0 by media CO₂ level or 1M sodium carbonate.

The permeate was collected at the same rate as the perfusion rate. Theperfusion rate increased gradually from 0.5 to 1.0 working volume/dayover the 15 day run. During the cultivation period, samples were takendaily, and cell counts, cell viability and metabolites were measured.Viable cell density, viability, titer and other measurements were madeas described above. Antibodies from bioreactor samples were firstpurified by affinity protein A MediaScout MiniChrom column (ATOLL,Weingarten, Germany) before the high mannose assay. The purified proteinwas digested with Endo H and denatured as described above. Thepercentage of NGHC from the digested samples and the controls weredetermined using ProteomeLab PA800 Capillary Electrophoresis system(Beckman Coulter, Brea, Calif.), and two NGHC values were used todetermine the HM % of the recombinant antibody.

FIGS. 4A-D show the time course for viable cell density, viability,antibody titer and % HM from the three tested bioreactors. For the cellculture perfused with the media having an M/H ratio of 0.94, the VCDreached 55×10⁶ cells/mL with 78% viability and the adjusted titer of14.3 g/L, which were all equivalent to the control. The metabolicprofiles are shown in FIGS. 5A-5D.

Cell growth, viability and titer were all slightly lower for M/H ratioof 0.5. This is likely due to an operational deviation for thiscondition. A base dump deviation at day 3 in the reactor with M/H ratioof 0.5 showed elevated osmolarity for 12 hours. The high osmolaritymight contribute to the slightly low VCD, viability and antibody titercompared to other two conditions FIGS. 4A-4C.

For HM content, the three test conditions showed different dynamicprofiles. The control had HM of 12% at day 6 and increased gradually to15% at day 15. When M/H ratio was 0.5, the HM was also 12% at day 6, butincreased two-fold to 24% at day 15. Interestingly, the culture grown inthe media having an M/H ratio of 0.94 was initially produced at a highHM of 26% and reached 38% at day 9 and plateaued. This indicates HMupregulation by mannose may exhibit a cap limitation effect.

These results indicate that the HM modulation effect is applicable atlarger scale. This effect is also predictable with an R-squared value of0.98 (FIG. 6). A good correlation was seen between the mock perfusionand bioreactor results. FIG. 6 compares the HM modulation effect betweenCell line A in the mock perfusion and at Day 15 of the bioreactor run.The slops of the trend lines are 22.5 and 23.8 for mock perfusion andbioreactor, respectively, demonstrating the correlation was scaleindependent.

Traditionally, when cells are cultured in flasks or on plates, physicalparameters and nutritional components are not well-controlled. Hence,scale variations regarding cell growth, protein production andglycosylation pattern are commonly observed in cell culture processes(Altamirano, et al. (2000) Biotechnol Prog 16, 69-75). In theseexperiments, it was surprising that there was a good correlation betweenthe mock perfusion assay and bioreactor scale cell cultures regardingthe HM modulation effects. This discovery makes the method even moreattractive as the bioreactor % HM becomes predictive. The high celldensity inoculation and daily media exchange which maintains a good pHcontrol in the mock perfusion might contribute to this correlation.

Example 3

To understand the overall glycan effect by mannose modulation,antibodies produced by Cell line A from the process described in Example2 were analyzed with a full glycan map assay usinghydrophilic-interaction liquid chromatography (HILIC). The purifiedantibodies were digested by N-glycosidase F (New England BioLabs,Ipswich, Mass.) at 37° C. for 2 hours to release the glycans. Thereleased glycans were labeled with 2-aminobenzoic acid and purifiedusing GlycoClean S cartridges (Prozyme, Heyward, Calif.) according tothe manufacturer's directions. The purified glycans were then desaltedand reconstituted in water for use in the assay. HILIC chromatographywas performed with a 100 mm×2.1 mm i.d BEH Glycan column using UPLC(Waters Corporation, Milford, Mass.) and the eluted glycans weredetected, identified, and qualified by a fluorescence detector based ondifferent elution times of different glycans.

As shown in the Table 3, certain glycans, including M3G0F, A2G1F, A2G2F,sialylated and aglycosylated forms, were not affected by different M/Hratios in the media.

TABLE 3 Glycan maps from bioreactor samples with various M/H ratios M/HHM M3G0F A2G0F A2G1F A2G2F Afucosylated Sialyated Unknown Aglycosylatedratio (%) (%) (%) (%) (%) (%) (%) glucan (%) (%) 0 13.4 5.9 63.1 9.0 0.83.8 0.6 2.2 2.2 0.5 19.8 5.5 50.5 11.9 1.3 4.8 0.8 3.4 1.6 0.94 30.6 5.940.4 8.9 0.8 5.6 0.7 5.0 2.9

A high M/H ratio increased the % HM and decreased the percent of A2G0F.The M/H ratio of 0.94 had the highest HM level of 30.6% and the lowestA2G0F level of 40.4%. In addition, an increased M/H ratio produced ahigher amount of afucosylated complex. A few existing unidentifiedglycan forms also increased to a small extent. The percentage of each HMspecies was also identified and compared (Table 4).

TABLE 4 Percentages of various HM species from bioreactor samples withvarious M/H ratios High M/H Mannose Man8 Man7 Man6 Man5 ratio (%) (%)(%) (%) (%) 0 13.4 0.4 0.4 0.6 12 0.5 19.8 1.2 1.8 2.4 14.4 0.94 30.62.5 3.1 5.9 19.2

It is known that GDP-mannose converts to GDP-fucose by GDP-mannose4,6-dehydratase (GMD). GDP-fucose is also a potent inhibitor of GMD(Becker and Lowe, (2003) Glycobiology 13, 41R-53R). It is speculatedthat high mannose concentration increases the influx of GDP-mannose,therefore, high intracellular GDP-fucose and fucosylation were expected.The increased afucosylated complex may be due to a strong feedbackinhibition of GDP-fucose to GMD or by unidentified mechanisms. Thegalactosylation and sialylation remain similar upon the addition ofmannose in the culture media. The decrease of A2G0F correlates with HMincrease. These results illustrate that only the early glycosylationpathway (before A2G0F) is majorly affected.

With an increase in M/H ratio, the levels of Man8, Man7, Man6 and Man5glycans were all significantly increased when compared to the control.Man5 was found to be the major HM species in all the three conditions;however Man6 to Man8 appeared to increase significantly at higher M/Hratios (Table 4). Considering antibody glycan processing, these resultssuggest that high M/H ratio slows down early glycan processing steps,leading to the elevated level of all HM species. With an increase of H/Mratio, more primitive high mannose species were found in the producedantibodies.

The HM species accumulation profile further suggests that twoglycosylation steps, HM trimming by α-mannosidase I (α-MAN I) and GluNAcaddition by GluNAc transferase (GnT1), are inhibited, increasing the HM% (FIG. 7). The first step for a cell to utilize intracellular mannoseis to convert mannose to mannose-6-phosphate, a key metabolite bridgingenergy generation (glycolysis) and N-glycan biosynthesis (Alton et al.,(1998) Glycobiology 8, 285-95). Elevated intracellular M6P level hasbeen demonstrated in mouse embryonic fibroblasts when 10 mM of mannosewas supplemented in the media (Gao et al., (2011) Mol Biol Cell 22,2994-3009). Therefore, it is reasonable to theorize that elevated M6Plevel should also be found in high M/H ratio culture, suggesting apossible flux increase for N-glycan biosynthesis. As illustrated in FIG.7, three pathways, GDP-mannose biosynthesis, early glycosylation andGluNAc biosynthesis, may involve in HM upregulation in this study.Increased levels of primitive HM species suggest that presence ofmannose of its intermediates (M6P, M1P or GDP-Mannose) may directly orindirectly inhibit α-mannosidase I activity. High Man5 levels alsosuggest GluNAc pathway might also be involved. However, our data showsthat the elevated Man5 level is not related to GDP-GluNAc limitation dueto glucose limitation effect (Table 5). Instead, it is more likely thatmannose or its intermediates affect GDP-GluNAc transportation or GnT1activity.

TABLE 5 % HM, residual glucose concentration in the media for Cell lineA High Residual Mannose Glucose Media (%) (g/L) G12M0 12.0 4.15 G9M316.0 2.86 G6M6 21.0 1.35 G3M9 27.3 0.97 G0M12 33.3 0.16

What is claimed is:
 1. A method for producing a denosumab composition ina CHO cell culture process comprising; establishing a CHO cell culturein a bioreactor with a cell culture media that does not contain mannose,said CHO cell culture containing CHO cells genetically engineered toexpress denosumab; growing the CHO cells during a growth phase with acell culture media that does not contain mannose; maintaining aproduction phase in the CHO cell culture with a feed medium comprisingmannose, wherein the mannose to total hexose ratio in the feed medium isgreater than 0 but less than 1.0; and recovering the denosumabcomposition from the cell culture.
 2. The method according to claim 1,wherein the high mannose glycoforms of denosumab in said denosumabcomposition are about 12% to about 30% of said composition.
 3. Themethod according to claim 1, wherein the high mannose glycoforms ofdenosumab in said denosumab composition are about 5% to about 21% ofsaid composition.
 4. The method according to claim 1, wherein the feedmedia is added by a bolus feed.
 5. The method according to claim 1,wherein the feed media is added by perfusion.
 6. The method according toclaim 1, wherein further comprising a step of purifying the denosumabcomposition.
 7. The method according to claim 1, wherein the purifieddenosumab composition is formulated into a pharmaceutically acceptableformulation.